The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.
The regulation of gene expression and cellular function in cells is controlled at many levels, including the regulation of the extent of chromatin structure, epigenetic control, transcriptional initiation and control of the rate thereof, messenger RNA (mRNA) transcript processing and modification, mRNA transport, mRNA transcript stability, translational initiation, control of transcript levels by small non-coding RNAs, post-translational modification, protein transport, and control of protein stability.
Antisense RNA (aRNA) and RNA interference (RNAi) technologies are well established tools for regulating gene expression through steric hindrance of translation and mRNA transcript degradation respectively. RNAi involves the introduction of short interfering RNA (siRNA) or microRNA (miRNA) into a cell, followed by the activation of RNAi cellular machinery and cleavage of a target messenger RNA (mRNA) transcript by RNA-induced silencing complex (RISC). However, the design of functional siRNA that is appropriately recognised by RNAi cellular machinery is highly complex and subject to various constraints (Tuschl, T. et al. (1999) Genes & Dev 13: 3191-3197). The siRNA must be 19-21 nucleotides in length and have a 2-nt 3′ overhang. The siRNA must exhibit limited G/C content and avoid consecutive stretches of the same base. Furthermore, the selectivity of strand loading into the RISC complex depends on the differential thermodynamic stabilities of the two ends of an siRNA duplex (Schwarz, S. D. et al. (2003) Cell 115:199-208), the less thermodynamically stable end being favoured for binding.
The design of pre-miRNA or miRNA to be processed into siRNA is further complicated by the requirement of secondary structural elements such as imperfectly base-paired stem regions flanked by free 5′ and 3′ ends and an unpaired loop region (Lund, E. and Dahlberg, J. E. (2006) Regulatory RNAs, Volume 71 of Cold Spring Harbor symposia on quantitative biology, CSHL Press). Thus, the constraints within which siRNA and miRNA molecules must be designed not only make the production of these molecules both challenging and complex but also limit the number and sequence of potential mRNA targets.
Despite the purported specificity of RNAi and aRNA technology for specific mRNA targets, cross-hybridization and non-specific binding can occur. In addition to the possibility for cross-hybridization of the antisense strand of siRNA to different mRNAs, siRNAs have demonstrated undesirable binding to various proteins (Bruckner, I. & Tremblay, G. A. (2000) Biochemistry 39: 11463-11466) causing significant nonspecific effects (Stein, C. A. (1995) Nat Med 1: 1119-1121). Moreover, the binding to affinity of siRNA-mediated binding of activated RISC to target mRNA (RNA-RNA interaction) is limited to that provided by canonical Watson and Crick base pairing, with guanine-cytosine interactions restricted to the expected three intermolecular hydrogen bonds, and adenine-uracil or uracil-guanine to the expected two intermolecular hydrogen bonds. Further disadvantages of RNAi technology include the need for transfection reagents or delivery vehicles, low and variable transfection efficiency sometimes necessitating the use of multiple transfection steps, partial and transient gene suppression effects, dependence upon processing by RNAi machinery, the limitation of mechanism to mRNA transcript degradation, and undesirable siRNA hairpin formation. siRNA is also known to be a potent activator of the mammalian innate immune system or IFN response (Judge, A, et al. (2008) Human Gene Therapy. (2008) 19: 111-124). The use of siRNA has been reported to cause an undesirable stimulation of immune activity and inflammatory response which may be further potentiated by the use of delivery vehicles, resulting in significant side effects due to excessive cytokine release and associated inflammatory syndromes. The potential for siRNA-based drugs to be rendered immunogenic is thus a cause for concern and has implications for both the development of siRNA-based drugs and the interpretation of gene-silencing effects elicited by siRNA (Judge, supra).
Alternatives to RNAi include the use of naturally occurring RNA-binding proteins which have been found to play essential roles in the regulation of gene expression. However, the modes by which such proteins bind RNA are idiosyncratic, restricted to sequence specific interactions, and difficult to predict so that their general use in biotechnological and medical applications is restricted. Information on the physiological targets of many RNA-proteins is limited and the binding of most RNA-proteins to their targets is reported to be idiosyncratic or require a combination of sequence and structural features such that their binding cannot be generally applied to other targets.
The PUF family of proteins (Drosophila Pumilio (Pum) and C. elegans FBF (fem-3 binding factor)) are an evolutionarily conserved family of RNA-binding proteins including Drosophila Pumilio and Caenorhabditis elegans FBF (for a review see Spassov, D. S. & Jurecic, R. (2003) IUBMB Life, 55: 359-366). PUF proteins contain an RNA-binding domain, known as the PUF domain or the Pumilio homology domain (PUM-HD), typically composed of eight tandem imperfect repeats of 36 amino acids plus conserved N and C-terminal flanking regions, aligned in tandem to form an extended curved arc-like molecule (Edwards, T. A. et al. (2001) Cell 105: 281-289). Target RNA binds to the inner concave surface of the protein, each of the eight repeats contacting a separate RNA base via three conserved amino acid residues positioned in the middle of the repeats (Wang, X. et al. (2002) Cell 110: 501-512). PUF proteins regulate RNA stability and translation by binding to specific sequences, such as the nanos response element (NRE), that are most often found in 3′ untranslated regions of target mRNAs (Gupta, supra). The PUM 1 NRE sequence is composed only of adenine, guanine or uracil.
The modular nature of the PUF-RNA interaction has been used to rationally engineer the binding specificity of PUF domains (Cheong, C. G. & Hall, T. M. (2006) PNAS 103: 13635-13639; Wang, X. et al (2002) Cell 110: 501-512). However, only the successful design of PUF domains with repeats that recognize adenine, guanine or uracil have been reported to date (Cheong, supra; Wang, supra). The specificity of individual repeats recognizing adenine, guanine or uracil were respectively switched by mutating only the positions that make contacts with the Watson-Crick edge of the base, providing engineered PUF domains capable of recognising endogenous RNA sequences composed of adenine, guanine or uracil (Wang, Y. Et al (2009) Nat Methods 6: 825-830; Tilsner, J. et al (2009) Plant 57: 758-770; Ozawa, T. et al (2007) Nat Methods 4: 413-419). Most of the known naturally occurring target sequences of PUM1, such as the NRE sequence, are composed of only adenine, guanine or uracil. However, despite focussed attempts to engineer PUF domains, the use of PUF domains designed with repeats that recognize these bases has remained substantially limited to sequences composed only of adenine, guanine or uracil.
There thus exists is a continued need for alternative methods for the specific regulation of gene expression and for agents for use therefor.